Target of anticancer agent

ABSTRACT

To provide a mutant Rad51 paralog gene, wherein a protein encoded thereby shows an activity for enhancing sensitivity of a cell to a DNA-damaging factor; a mutant Rad51 paralog peptide showing the activity; a transformed cell having the gene; a screening method for a drug having a DNA-damaging action, comprising contacting a test substance with the transformed cell, and evaluating a response of the cell; and a screening method for a controlling agent for DNA repair, comprising contacting a test substance with a transformed cell having the Rad51 paralog gene, and evaluating a homologous recombination repair capacity. According to the present invention, there is enabled a screening of a novel anticancer agent which allows a more efficient therapy for a cancer, wherein the agent is an agent capable of enhancing the sensitivity of a cell to an anticancer agent comprising a DNA-damaging factor or an agent having a DNA-damaging action.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP00/04739 which has an Internationalfiling date of Jul. 14, 2000, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a target for an anticancer agent and atechnique using the same, which are useful for the treatment of cancer.

BACKGROUND ART

Double-strand DNA breakages occur frequently during DNA repair and areproduced by ionizing radiation and certain chemicals [Haber, J. E.,Trends Biochem. Sci., 24, 271–275 (1999)]. A single unrepaireddouble-strand DNA breakage is believed to cause cell death in yeast andvertebrate cells [Bennett, C. B. et al., Proc. Natl. Acad. Sci. USA, 90,5613–5617; Huang, L. C. et al., Proc. Natl. Acad. Sci. USA, 93,4827–4832 (1996)].

In yeast, homologous recombination repair is a major DNA breakage repairpathway. The above-mentioned homologous recombination repair pathway hasbeen conserved from yeast to human [Shinohara, A. et al., Nat. Genet.,4, 239–243 (1993); Siede, W. et al., Genetics, 142, 91–102 (1996);Boulton, S. J. et al., Nucleic Acids Res., 24, 4639–4648 (1996a);Boulton, S. J. et al., EMBO J., 15, 5093–5103 (1996b); Bezzubova, O. Y.et al., Cell, 89, 185–193 (1997); Essers, J. et al., Cell, 89, 195–204(1997); Thompson, L. H. et al., Biochimie, 81, 87–105 (1999)].

Presently, the analysis of radiosensitive yeast mutants has revealednumbers of key genes involved in the homologous recombination repair.The genes include, for instance, the Rad52 epistasis group and the like[Shinohara, A. et al., Trends Biochem. Sci., 20, 387–391 (1995);Baumann, P. et al., Trends Biochem. Sci., 23, 247–251; Kanaar, R. etal., Trends Cell Biol., 8, 483–489 (1998)].

Among the members of the RAD52 epistasis group, the structure andfunction of Rad51 have been conserved to a remarkable degree among alleukaryotes. The above-mentioned Rad51 structurally and functionallyresembles Escherichia coli recombination protein RecA [reviewed inKowalczykowski, S. C. et al., Experientia, 50, 204–15 (1994)]. Lethalityof Rad51-deficient cells [Tsuzuki. T et al., Proc. Natl. Acad. Sci. USA,93, 6236–6240 (1996); Lim, D. S. et al., Mol. Cell Biol., 16, 7133–7143(1996); Sonoda. E. et al., EMBO. J., 17, 598–608 (1998)] has suggestedthat Rad51 plays a central role in the homologous recombination repairin vertebrate cells [Bezzubova, O. Y. et al., Cell, 89, 185–193 (1997);the above-mentioned Essers et al. (1997); Rijkers, T. et al., Mol. CellBiol., 18, 6423–6429 (1998); Yamaguchi-Iwai, Y. et al., Mol. Cell.Biol., 18, 6430–6435 (1998)].

However, many of the detailed functions and roles for other Rad52epistasis groups are yet unrevealed at the current situation.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a target molecule (anucleic acid, a peptide) which is useful for developing an anticanceragent having an action for enhancing the sensitivity of a cell to aDNA-damaging factor. Also, an object of the present invention is toprovide a screening method for an anticancer agent having a DNA-damagingaction, and a cell which is suitable for the screening method. Further,an object of the present invention is to provide a screening method fora controlling agent for DNA repair, which can be applied to ananticancer agent, a prophylactic agent for cancer, or the like.

Concretely, the present invention relates to:

[1] a mutant Rad51 paralog gene having substitution, deletion, insertionor addition of at least one base in:

(A) a nucleotide sequence selected from the group consisting of SEQ IDNOs 1, 3 and 5; or

(B) a nucleotide sequence different from the nucleic acid of the (A)above via degeneracy,

wherein a protein encoded thereby shows an activity for enhancingsensitivity of a cell to a DNA-damaging factor;

[2] a mutant Rad51 paralog peptide having substitution, deletion,insertion or addition of at least one amino acid residue in an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 4 and6, wherein the mutant Rad51 paralog peptide shows an activity forenhancing sensitivity of a cell to a DNA-damaging factor;[3] a transformed cell having the gene of the [1] above;[4] a screening method for a drug having a DNA-damaging action,comprising the steps of:(1) contacting a test substance with the transformed cell of the [3]above; and(2) evaluating a response of the cell obtained in step (1); and[5] a screening method for a controlling agent for DNA repair,comprising the steps of:(I) contacting a test substance with a transformed cell having a nucleicacid comprising (A) a nucleotide sequence selected from the groupconsisting of SEQ ID NOs 1, 3 and 5; or(B) a nucleotide sequence different from the nucleic acid of the (A)above via degeneracy; and(II) evaluating a homologous recombination repair capacity in the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the results for evaluating lowered viabilityin mutant cultures. The panel (A) shows the results in which the levelof spontaneous cell death was evaluated by flow cytometric analysisusing propidium iodide (PI) uptake (Y axis) and forward scatter showinga cell size (X axis). Numbers show the percentage of dead (PI-bright andPI-dim/small) cells, and the solid line separates live cells from deadcells. In the panel (B), bars represent the level of spontaneous celldeath in indicated genotypes.

FIG. 2 is a diagram showing the levels of sister chromatid exchange percell before and after MMC treatment. One-hundred and fifty cells wereanalyzed in each preparation. Error bars represent SD.

FIG. 3 are graphs each showing the sensitivity of knockout cell lines toDNA-damaging factors. The panel (A) shows survival curves aftertreatments with γ-ray irradiation and MMC. Sensitivity data of rad54 andrad51b cells were previously described (Tanaka et al., submitted). Thepanel (B) shows partial enhancement of cisplatin sensitivity in knockoutmutants by overexpression of human Rad51. Data shown are representativeof at least two independent experiments. The panel (C) shows the resultsof Western blot analysis of human Rad51 transformants derived fromknockout mutants. The transformants have much higher steady-state levelsof cDNA-derived human Rad51 than endogenous Rad51.

FIG. 4 is a photograph showing the results of induction of Rad51 foci bygenotoxic treatments. Immunofluorescence of Rad51 nuclear foci afterγ-ray irradiation (8 Gy) (A to E) or MMC treatment (500 ng/ml, 1 hour)(F to J) was visualized as previously described [Yamaguchi-Iwai et al.(1998)]. In the figure, A and F are wild-type; B and G are rad51c; C andH are rad51d; D and I are xrcc2; E and J are xrcc3, respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is based on a surprising finding by the presentinventors that a Rad51 paralog peptide, concretely a cell deficient ineach of Rad51B, Rad51C and Rad51D genes, shows high sensitivity to aDNA-damaging factor, concretely cisplatin, mitomycin C or the like.

In view of the above finding, there are provided an application toscreening of a drug having a higher anticancer action when the DNAdamaging factor, for instance, cisplatin, mitomycin C or the like isused as an anticancer agent, and an application to screening of a drugcapable of controlling an action of Rad51 paralog peptide.

Also, the present invention is based on an excellent technical idea thatthe proliferating cells such as cancer are led to death by suppressionof the homologous recombination.

In the present specification, the term “paralog” is used to refer to agroup of plural genes having high structural resemblance with each otherin a single species, wherein the genes are generated by a duplication ofsingle ancestor gene, or protein products thereof.

The results in Examples set forth below and other studies [Johnson, R.D. et al., Nature, 401, 397–399 (1999); Pierce, A. J. et al., GenesDev., 13, 2633–2638 (1999) and the like] show that all five kinds of theRad51 paralogs are important for homologous recombination repair invertebrate cells. Remarkably, mutant clones for each Rad51 paralog arephenotypically similar, suggesting that each protein is vitally involvedin a particular process in homologous recombination repair. On the otherhand, two hybrid analyses suggest that each Rad51 paralog appears tohave different interacting partners within the family and together canform a single complex [reviewed in Dosanjh, M. K. et al., Nucleic AcidsRes., 26, 1179–1184 (1998); Liu, N. et al., Mol. Cell, 1, 783–793(1998); the above-mentioned Thompson et al., (1999)]. These biochemicalresults combined with the genetic data by the present inventors areconsistent with the action of the Rad51 paralogs as a single functionalunit during homologous recombination repair.

The data given in Examples set forth below have suggested that yeastRad51 paralogs, Rad55 and Rad57 are involved in the formation ofnucleoprotein filaments involving Rad51. First, protein interactionsbetween Rad51 and Rad55 and those between Rad55 and Rad57 suggest thatthese molecules act in multiprotein complexes. Second, the repairdefects of rad55/57 mutants are partially suppressed by theoverexpression of Rad51 [Hays, S. L. et al., Proc. Natl. Acad. Sci. USA,92, 6925–6929 (1995); Johnson, R. D. et al., Mol. Cell Biol., 15,4843–4850 (1995)]. Third, biochemical analysis points out the Rad55/57heterodimer acting as a cofactor for promoting an assembly ofRad51-ssDNA nucleoprotein filaments in the presence of RPA [Sung, P.,Genes Dev., 11, 1111–1121 (1997)]. These results support the idea thatRad55 and Rad57 are involved in homologous recombination repair byforming a complex which interacts with Rad51 to promote the formation ofRad51 nucleoprotein filaments.

The functional interactions between Rad51 and Rad51 paralogs have beeninvestigated in the genetic system of the present invention. Theoverexpression of human Rad51 at least partially normalized thedeficiency of each Rad51-paralog mutant in repairing genomic damage byγ-rays, cisplatin, and MMC. This observation implies that each Rad51paralog participates in homologous recombination repair by facilitatingthe function of Rad51, as does Rad55/Rad57 in yeast. In addition,defective Rad51 focus formation in Rad51-paralog-mutants suggests thatthe Rad51 paralogs can promote the assembly of Rad51 at DNA lesions. Asimilar situation applies to yeast, in which case mutations in Rad55 andRad57 prevent the appearance of Rad51 foci during meiosis [Gaisor, S. L.et al., Genes Dev., 12, 2208–2221 (1998)]. These observational resultsshow that the Rad51 paralogs facilitate the function of Rad51 bypromoting the assembly of Rad51 in nucleoprotein filaments.

While the data in Examples set forth below suggest that the Rad51paralogs can be involved in homologous recombination by association withRad51, Kurumizaka et al. (in preparation for contribution of an article)recently found that a complex composed of Rad51C and XRCC3 shows astrong homologous pairing activity, but not showing a branch migrationactivity, in the absence of Rad51 in vitro. This in vitro activity mightpromote intragenic double-stranded DNA breakage-mediated homologousrecombination repair in vivo, the level of which was reduced by 25- to100-fold in mammalian XRCC2- and XRCC3-deficient cells and was notrestored by transient transfection with human Rad51 [the above-mentionedJohnson et al. (1999); the above-mentioned Pierce et al. (1999)]. Thisdrastic reduction in intragenic homologous recombination repairefficiencies was in marked contrast with only a few fold reduction inradiation resistance to γ-rays in late-S-phase [Cheong, N. et al.,Mutat. Res., 314, 77–85 (1994)]. In this case, induced double-strandedDNA breakages on one chromatid should be repaired by homologousrecombination with the other intact sister chromatid [Takata, M. et al.,EMBO J., 17, 5497–5508 (1998)]. These observational results show thepossibilities that some Rad51 paralogs are involved in an intragenichomologous recombination subpathway that does not require Rad51. Giventhat the Rad51 paralogs, but not Rad51, are expressed in somenondividing cells (e.g. all Rad51 paralogs are expressed in the brain),the Rad51 paralogs are possible to play a role in intragenic homologousrecombination repair in resting cells of vertebrates.

According to Examples set forth below, it is shown that DNA damageinduces Rad51 foci in a Rad51 paralog-dependent manner, and that allRad51-paralog mutants are highly sensitive to cisplatin. Thus, eachRad51 paralog plays an important role in the response to this clinicallyimportant drug. Since XRCC2-deficient cells are shown to exhibit adrastic increase in chromosomal breakages following exposure to MMC[Tucker, J. D. et al., Mutat. Res., 254, 143–152 (1991)], the formationof unrepaired double-stranded DNA breakages during abortive cross-linkrepair most likely explains the extremely high sensitivity ofXRCC2-deficient cells to MMC.

The occurrence of homologous recombination repair during the normalmeiotic cell cycle is suggested by the appearance of Rad51 foci in Sphase and by spontaneous sister chromatid exchange. The sister chromatidexchanges are at least partially mediated by homologous recombinationrepair and occur at a frequency of about 3 exchanges per single cellcycle in a mammalian cell [Sonoda. et al., Mol. Cell. Biol., 19,5166–5169 (1999)]. In addition, the presence of excessive chromosomebreakages in rad51 avian-cell mutant and mre11 avian-cell mutantindicates that homologous recombination repair plays an essential rolein repairing potentially lethal chromosomal breakages that are likely tooccur during DNA replication [Sonoda et al. (1998); Yamaguchi-Iwai etal., EMBO J., 18, 6619–6629 (1999)]. Thus, defective homologousrecombination repair exhibits a phenotype similar to that of the humanchromosome instability syndromes, such as Bloom syndrome, Fanconi anemiaand ataxia telangiectasia. These all show an increased incidence ofcancer [reviewed in Meyn, M. S., Curr. Top. Microbiol. Immunol., 221,71–148 (1997)]. Given that Rad51 paralogs also function as tumorsuppressor genes by maintaining the integrity of chromosomes, it isdesirable to screen for mutations in these loci in various tumors.Indeed, chromosome translocation breakpoints within Rad51B at position14q23–24 have been recently found in uterine leiomyomas [Schoenmakers,E. F. et al., Cancer Res., 59, 19–23 (1999)].

The Brca2 cancer susceptibility protein is associated with Rad51 inmitotic cells and meiotic cells [Chen, J. et al., Mol. Cell, 2, 317–328(1998a); Chen, J. et al., Proc. Natl. Acad. Sci., USA, 95, 5287–5292(1998b)], suggesting a direct role of Brca2 in the homologousrecombination repair. It is noteworthy that human Brca2-truncated mutantcells and murine Brca2-truncated mutant cells exhibit phenotypes:elevated spontaneous chromosomal aberrations [Patel, K. J. et al., Mol.Cell, 1, 347–357 (1998)], sensitivity to MMC [the above-mentioned Patelet al. (1998)], and defective Rad51 focus formation [Yuan, S. S. et al.,Cancer Res., 59, 3547–3551 (1999)], which are remarkably similar to theRad51-paralog mutants. Thus, Brca2 is possible to participate in theformation of a complex involving the Rad51 paralogs, and acts as acofactor of Rad51 during the homologous recombination repair. Inaddition to the presence of Brca2 homologs in vertebrates but not inyeast, the presence of five kinds of Rad51 paralogs in vertebratesinstead of only two kinds in yeast (Rad55 and Rad57) shows that theassembly of Rad51 during homologous recombination repair is regulated ina more complicated manner in vertebrate cells. Indeed, althoughhomologous recombination repair occurs efficiently in the G1 phase indiploid yeast [Kadyk, L. C. et al., Genetics, 132, 387–402 (1992)],there was no induction of Rad51 focus formation by ionizing radiation inthe G1 phase in CHO hamster cells [Bishop, D. K. et al., J. Biol. Chem.,273, 21482–21488 (1998)]. This finding shows that the assembly of Rad51is possible to be actively suppressed in the G1 phase to prevent geneconversion between homologous chromosomes, which would result in loss ofheterozygosity.

The above-mentioned Rad51 paralog peptide (hereinafter simply referredto “Rad51 paralog” in some cases) includes, for instance, Rad55 andRad57 from Saccharomyces cerevisiae; and XRCC2 [Cartwright, R. et al.,Nucleic Acids Res., 26, 3084–3089 (1998); Liu, N. et al., Mol. Cell, 1,783–793 (1998)], XRCC3 [Tebbs, R. S. et al., Proc. Natl. Acad. Sci. USA,92, 6354–6358 (1995); the above-mentioned Liu et al. (1998)], Rad51B[Albala, J. S. et al., Genomics, 46, 476–479 (1997); Rice, M. C. et al.,Proc. Natl. Acad. Sci. USA, 94, 7417–7422 (1997); Cartwright, R. et al.,Nucleic Acids Res., 26, 1653–1659 (1998)], Rad51C [the above-mentionedDosanjh, M. K. et al. (1998)], and Rad51D [Pittman, D. L. et al.,Genomics, 49, 103–111 (1998); Cartwright et al., Nucleic Acids Res., 26,1653–1659 (1998); Kawabata, M. et al., Biochem. Biophys. Res. Commun.,1398, 353–358 (1998)], which are derived from vertebrates.

Among them, the vertebrate-derived, especially the five kinds ofhuman-derived Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C and Rad51D)have only 20–30% identity with human Rad51 and show only less than 30%homology to each other. Also, the above-mentioned five kinds of humanRad51 paralogs show only less than 30% homology to yeast Rad55 and Rad57[reviewed in Thacker, Trends Genet., 15, 166–168 (1999)].

Unlike Rad51, none of the above-mentioned Rad51 paralog appears tointeract with itself as in the case of yeast Rad55 and Rad57 [theabove-mentioned Thompson et al. (1999)]. It is suggested thatoverexpression of Rad51 partially suppresses the DNA repair defect inrad55 and rad57 mutant yeast strains, whereby the Rad55 and Rad57functionally cooperate with Rad51. This idea is supported by physicalinteractions between Rad51 and Rad55 and physical interactions betweenRad55 and Rad57 [the above-mentioned Hays, S. L. et al. (1995); theabove-mentioned Johnson, R. D. et al. (1995); Sung, P. (1997)].Similarly, physical interactions occur between human Rad51 and XRCC3,between XRCC3 and Rad51C, between Rad51B and Rad51C, and between Rad51Cand Rad51D [reviewed in the above-mentioned Thompson et al. (1999)].These observation results show that Rad51 paralogs form a functionalcomplex and cooperate with Rad51, in the same manner as in the yeastRad55 and Rad57 proteins.

1. Mutant Rad51 Paralog Gene of the Present Invention

According to the present invention, there is provided a mutant Rad51paralog gene, wherein a protein encoded thereby shows an activity forenhancing sensitivity of a cell to a DNA-damaging factor.

The mutant Rad51 paralog gene of the present invention includes a genehaving substitution, deletion, insertion or addition of at least onebase in:

(A) a nucleotide sequence selected from the group consisting of SEQ IDNOs: 1, 3 and 5; or

(B) a nucleotide sequence different from the nucleic acid of the (A)above via degeneracy,

wherein a protein encoded thereby shows an activity for enhancingsensitivity of a cell to a DNA-damaging factor.

The mutant Rad51 paralog gene of the present invention is one in whichits encoded protein shows an activity for enhancing sensitivity of acell to a DNA-damaging factor, especially has lowered or diminishedhomologous recombination repair capability. Therefore, when aDNA-damaging factor, for instance, cisplatin, mitomycin C or the like isused as an anticancer agent, the paralog gene is especially useful inthe application for screening of a drug having a higher anticanceraction.

Here, the above-mentioned nucleotide sequences of SEQ ID NOs: 1, 3 and 5are sequences of a gene encoding Rad51B, a gene encoding Rad51C and agene encoding Rad51D. Incidentally, genes encoding Rad51B, Rad51C andRad51D may be referred to rad51b, rad51c and rad51d, respectively, insome cases.

The above-mentioned protein includes a mutant peptide which is deficientof the function of Rad51B, Rad51C and Rad51D.

More concretely, the above-mentioned mutant Rad51 paralog gene includes:

(a) a nucleotide sequence in which a region of 463rd to 573rd bases isdeleted or substituted with a sequence of a marker gene in thenucleotide sequence of SEQ ID NO: 1,

(b) a nucleotide sequence in which a region of 628th to 747th bases isdeleted or substituted with a sequence of a marker gene in thenucleotide sequence of SEQ ID NO: 3, and

(c) a nucleotide sequence in which a region of 536th to 583rd bases isdeleted or substituted with a sequence of a marker gene in thenucleotide sequence of SEQ ID NO: 5,

wherein a protein encoded thereby shows an activity for enhancingsensitivity of a cell to a DNA-damaging factor.

In the present invention, the mutant Rad51 paralog gene is not limitedto the gene having any of the sequences of the (a) to (c) above. Forinstance, there is included a gene having a nucleotide sequence havingsubstitution, deletion, insertion or addition of at least one base inany of SEQ ID NOs: 1, 3 and 5 so that the encoded protein is deficientof homologous recombination repair capacity, wherein a protein encodedthereby shows an activity for enhancing sensitivity of a cell to aDNA-damaging factor.

Here, substitution, deletion, insertion or addition can be easilyobtained by one of ordinary skill in the art by such a method asconventional site-directed mutagenesis method or PCR method. Thetechniques are described in textbooks, for instance, Molecular Cloning:A Laboratory Manual, 2nd Ed. [Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press (1989)], and the like. Thenumber of substitution, deletion, insertion or addition can be properlyselected within the range in which the protein encoded by the resultinggene shows an activity for enhancing sensitivity of a cell to aDNA-damaging factor.

The above-mentioned mutant Rad51 paralog gene can be obtained by:

1) introducing a desired mutation (substitution, deletion, insertion andaddition) into the nucleotide sequence of SEQ ID NOs: 1, 3 and 5mentioned above to give a mutant gene, and

2) evaluating an expression product of the resulting mutant gene for anactivity for enhancing sensitivity of a cell to a DNA-damaging factor,optionally homologous recombination repair capacity.

Here, the phrase “activity for enhancing sensitivity of a cell to aDNA-damaging factor” can be evaluated as described, for instance, inExample 3. Also, the term “homologous recombination repair capacity” canbe evaluated by analyses such as the analysis for chromosomalaberrations, the analysis for sister chromatid exchanges, the analysisof targeted integration frequencies and the like as described in Example2.

In the present invention, the DNA-damaging factor includes irradiationrays (γ-rays and the like), mitomycin C and cisplatin. Among them,mitomycin C and cisplatin possess a characteristic of forming across-link between the double strand of the DNA molecule, therebyspecifically inhibiting only the DNA biosynthesis selectively, so thatthese substances can be used as an anticancer agent. The mutant Rad51paralog gene of the present invention via the expression exhibitsexcellent effects such that the sensitivity of a cell to an anticanceragent comprising the DNA-damaging factor as mentioned above can beimproved.

The above-mentioned marker gene may be any of those which are deficientof the function of the Rad51 paralog gene and can facilitate selectionof the desired mutant Rad51 paralog gene, and includes drug resistancegenes, for instance, puromycin resistance gene, neomycin resistancegene, histidinol resistance gene, Ecogpt gene, blastocitidin resistancegene, hygromycin resistance gene, and the like.

The mutant Rad51 paralog gene of the present invention serves to improvethe sensitivity of a cell to an anticancer agent comprising theabove-mentioned DNA-damaging factor by expressing its antisense strandin a tumor tissue to specifically control its gene product. Therefore,the mutant Rad51 paralog gene can be expected to be used as an agent forgene therapy, wherein the agent exhibits an anticancer action.

2. Mutant Rad51 Paralog Peptide of the Present Invention

There can be provided a mutant Rad51 paralog peptide by theabove-mentioned mutant Rad51 paralog gene. The mutant Rad51 paralogpeptide is also encompassed in the scope of the present invention. Themutant paralog peptide also includes a peptide having mutations inWalker motif, which is an active center of ATP hydrolyzing activity. Bythe mutations in this motif, there can be prepared a mutant paralogpeptide having dominant negative activity.

The mutant Rad51 paralog peptide of the present invention includes agene encoded by the above-mentioned mutant Rad51 paralog gene. Further,the mutant Rad51 paralog peptide of the present invention includes amutant Rad51 paralog peptide having substitution, deletion, insertion oraddition of at least one amino acid residue in an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 4 and 6, whereinthe mutant Rad51 paralog peptide shows an activity for enhancingsensitivity of a cell to a DNA-damaging factor.

Here, the amino acid sequences of SEQ ID NOs: 2, 4 and 6 mentioned aboveshow the sequences of Rad51B, Rad51C, and Rad51D, respectively.

Concretely, the above-mentioned mutant Rad51 paralog peptide includes apeptide having an amino acid sequence selected from the group consistingof:

(i) an amino acid sequence in which a region of 137th to 173rd aminoacids is deleted or substituted with a sequence of a marker peptide inthe amino acid sequence of SEQ ID NO: 2;

(ii) an amino acid sequence in which a region of 196th to 235th aminoacids is deleted or substituted with a sequence of a marker peptide inthe amino acid sequence of SEQ ID NO: 4; and

(iii) an amino acid sequence in which a region of 138th to 153rd aminoacids is deleted or substituted with a sequence of a marker peptide inthe amino acid sequence of SEQ ID NO: 6.

When the mutant Rad51 paralog peptide, especially Rad51B mutant peptide,Rad51C mutant peptide or Rad51D mutant peptide, is expressed in a cell,there is exhibited an excellent characteristic that the mutant peptidehas a high specificity to cisplatin as compared to that of a known Rad51mutant peptide.

The “activity for enhancing sensitivity of a cell to a DNA-damagingfactor” can be evaluated as described in, for instance, Example 3.

Since the mutant Rad51 paralog peptide of the present invention shows anactivity for enhancing sensitivity of a cell to a DNA-damaging factor,the mutant Rad51 paralog peptide is useful as a target for an anticanceragent comprising a DNA-damaging factor. Accordingly, there is expectedits application as an anticancer agent in combined use with theanticancer agent comprising a DNA-damaging factor.

Also, in the mutant Rad51 paralog peptide of the present invention, theencoded protein shows an activity for enhancing sensitivity of a cell toa DNA-damaging factor, and especially its homologous recombinationrepair capacity is lowered or diminished. Therefore, when a DNA-damagingfactor, for instance, cisplatin, mitomycin C or the like, is used as ananticancer agent, the mutant Rad51 paralog peptide is especially usefulfor an application to a screening of a drug having a higher anticanceraction.

3. Transformed Cell of the Present Invention

According to the mutant Rad51 paralog gene of the present invention,there is provided a transformed cell having the gene. The transformedcell is also encompassed in the scope of the present invention. Sincethe transformed cell of the present invention has the above-mentionedmutant Rad51 paralog gene, the sensitivity of a cell to a DNA-damagingfactor is further enhanced as compared to a normal cell. Therefore, thetransformed cell is useful for screening a drug having a DNAcross-linking ability comparable to cisplatin or the like.

The transformed cell of the present invention can be obtained byintroducing the mutant Rad51 paralog gene of (1) mentioned above into ahost cell.

A subject for a host cell is not only DT40 cell but also all the humancell lines. Among them, the above-mentioned DT40 cell is an excellentmodel for detailed functional analyses of Rad5 paralog because the DT40cells have more efficient homologous recombination repair capacity thanthat of mammalian cells [Buerstedde, J. M. et al., Cell, 67, 179–188(1991)], and gene targeting therefor is easy.

A means for introducing a gene into a host cell includes, for instance,calcium phosphate method, lipofection method, electroporation method andthe like. When a gene is introduced into a host cell, there may be useda recombinant vector resulting from incorporation of a mutant Rad51paralog gene into a vector depending upon the host cell used.

In a case where a marker gene exists on the mutant Rad51 paralog gene,the transformed cell can be sorted out by using the expression of themarker gene as an index. Also, in a case where a vector is used when agene is introduced into a host cell, the transformed cell can be sortedout by using expression of the marker gene on the vector as an index.

As to the culture of the transformed cell, appropriate conditions can beset depending upon the host cell. The culture conditions for thetransformed cell are not special ones, and are as follows: incubation ina Dulbecco's liquid medium containing an antibiotic, fetal bovine serumand avian serum with an incubator at 5% CO₂ and 37° C.

In the present specification, the transformed cells deficient in eachgene of the above-mentioned Rad51B, Rad51C and Rad51D are referred torad51b cell, rad51c cell, rad51d cell, respectively.

4. Screening Method of the Present Invention

According to the above-mentioned transformed cell, there can beperformed screening of a drug having a DNA-damaging action. The presentinvention also encompasses the screening method.

The screening method of a drug having a DNA-damaging action of thepresent invention comprises the steps of:

(1) contacting a test substance with the above-mentioned transformedcell; and

(2) evaluating a response of the cell obtained in the step (1).

In the screening method, the transformed cell having sensitivity of acell to a DNA-damaging factor. Therefore, according to the method, thereis exhibited an excellent effect such that a drug having aDNA-cross-linking ability comparable to cisplatin or the like can bescreened.

The test substance includes a compound, a peptide, an extract and thelike as desired.

In the step (1), the contact of the test substance with the transformedcell can be carried out by adding a test substance to a medium of thetransformed cell so as to have an appropriate concentration, andculturing the transformed cell. The culture conditions can beappropriately set depending upon the transformed cell.

In the step (2), the response of the cell can be evaluated by usingviability of the cell, chromosomal breakages, homologous recombination(targeted integration frequency) on the chromosomes, sister chromatidexchange, and the like as an index.

For instance, when in the transformed cell in the presence of a testsubstance, the level of the chromosomal breakages has been increased,the homologous recombination (targeted integration frequency) on thechromosomes has been lowered, and the level of the sister chromatidexchange is lowered, as compared to those in the absence of the testsubstance, the test substance can serve as an index of a drug havingDNA-damaging action. Also, in an appropriate host cell, the accuracy ofthe screening can be further increased by carrying out the sameprocedures as the evaluation in the transformed cell.

5. Screening Method for DNA Repair Controlling Agent

According to the findings of the present inventors, there can be furtherprovided a screening method for a DNA repair inhibitor.

According to the findings of the present inventors, since Rad51paralogs, especially Rad51B, Rad51C and Rad51D, are deeply involved inthe homologous recombination repair, a controlling agent for DNA repaircan be screened by evaluating the increase or decrease, or presence orabsence of the function of normal Rad51B, Rad51C or Rad51D. In addition,the above-mentioned Rad51B, Rad51C and Rad51D have an excellentcharacteristic of high specificity to cisplatin when the above-mentionedRad51B, Rad51C and Rad51D are expressed in the cell, as compared to aknown Rad51.

Concretely, the screening method for a controlling agent for DNA repairof the present invention comprises the steps of:

(I) contacting a test substance with a transformed cell having a nucleicacid having

(A) a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3 and 5; or

(B) a nucleotide sequence different from the nucleic acid of the (A)mentioned above via degeneracy; and

(II) evaluating a homologous recombination repair capacity in the cell.

The transformed cell having a nucleic acid having the (A) or (B)mentioned above can be prepared by the same technique as that for thetransformed cell described in section 3. above. For instance, atransformed cell can be obtained by incorporating a nucleic acid havingthe (A) or (B) mentioned above into an appropriate vector, andintroducing the resulting recombinant vector into an appropriate host.

In the step (I), the contact of the test substance with the transformedcell can be carried out by adding a test substance to a medium of thetransformed cell so as to have an appropriate concentration, andculturing the transformed cell. The culture conditions can beappropriately set depending upon the transformed cell.

In the step (II), the homologous recombination repair capacity can beevaluated by using chromosomal breakages, homologous recombination(targeted integration frequency) on the chromosomes, sister chromatidexchange, and the like as an index. For instance, the homologousrecombination repair capacity can be evaluated by the analysis of sisterchromatid exchange, the analysis of the targeted integration frequencyand the like as described in Example 2.

As to the index showing that the test substance is a controlling agentfor DNA repair, in the transformed cell in the presence of a testsubstance, when the homologous recombination (targeted integrationfrequency) on the chromosomes is lowered, and the level of the sisterchromatid exchange is lowered, as compared to those in the absence ofthe test substance, the test substance can serve as an index of havingDNA repair-suppressing capacity. On the other hand, in the transformedcell in the presence of a test substance, when the homologousrecombination (targeted integration frequency) on the chromosomes isincreased, and the level of the sister chromatid exchange is increased,as compared to those in the absence of the test substance, the testsubstance can serve as an index of having DNA repair-enhancing capacity.Also, in an appropriate host cell, the accuracy of the screening can befurther increased by carrying out the same procedures as the evaluationin the transformed cell.

The controlling agent for DNA repair obtained by the screening methodcan be utilized for controlling the DNA repair capacity. Concretely, ina case of a drug that lowers a DNA repair capacity, the drug forenhancing sensitivity of a cell to a DNA-damaging factor can beadministered in combination with an anticancer agent having aDNA-damaging action, represented by cisplatin, mitomycin C.

The present invention will be described in further detail by means ofExamples, without intending to limit the present invention to theseExamples.

EXAMPLE 1 Involvement of rad51 Gene in Cell Growth

(1) Preparation of rad51 Gene Targeting Construct

Each of Rad51 paralog defective mutant clones (rad51c mutant, rad51dmutant, xrcc2 mutant and xrcc3 mutant) shown in the panel (B) of FIG. 1were prepared from avian cell line DT40 [the above-mentioned Buersteddeet al. (1991)] as described below.

Genomic DNA fragments of the Rad51-related genes were isolated from DT40genomic DNA by long-range PCR with primers based on cDNA sequences.Next, the gene targeting constructs were prepared in accordance with thedescription of the above-mentioned Buerstedde et al. (1991) as follows:Concretely, both of two kinds (both sides) of several-kb genomic DNAfragments on both sides of the genomic DNA of the region to bedisrupted, which were isolated by the above-mentioned long-range PCRwere cloned in an appropriate vector. Next, a plasmid in which aselection marker was inserted between the two kinds of the DNAs wasprepared. Thereafter, a DNA resulting from cleavage of thevector-derived portion in the above-mentioned plasmid was introducedinto DT40 cells by electroporation using the resulting DNA as a genetargeting construct.

Regarding these constructs, gene targeting was performed so that anamino acid sequence corresponding to each of the published human genes:196th to 235th amino acids in Rad51C [the above-mentioned Dosanjh et al.(1998); GenBank Accession NO: AF029669], 138th to 153rd amino acids inRad51D [Cartwright, R. et al., Nucleic Acids Res., 26, 1633–1659 (1998);GenBank Accession NO: AF034956], 47th to 89th amino acids in XRCC2 [theabove-mentioned Liu et al. (1998); GenBank Accession NO: AF035587], and212th to 242nd amino acids in XRCC3 [the above-mentioned Tebbs et al.(1995); GenBank Accession NO: AF035586] was substituted with a selectionmarker (puromycin resistance gene or the like).

Next, the human Rad51 and Rad51 paralogs were incorporated into anexpression vector prepared on the basis of expression vector pAGS3carrying the avian β-actin promoter (prepared by Professor Kurasaki ofOsaka University, Faculty of Medicine), to give a recombinant vector.The resulting recombinant vector was transfected into DT40 cell line.The DNA transfection was carried out in accordance with the conditionsas described in the above-mentioned Buerstedde et al. (1991).Concretely, the DNA transfection was carried out under the conditionssuch that 50 μg of a gene targeting construct DNA was placed in PBS(−),that 10⁷ cells were suspended in 0.8 ml of the above-mentioned solution[PBS(−)], that the suspension was set in Gene pulser of Bio-Rad, andthat electric pulses were applied to the cells at 25 μF and 550 V.

(2) Culture of Mutant Clones

For the mutant clone obtained in (1) above, the cell culture was carriedout in accordance with the conditions as described in Buerstedde et al.(1991), supra. Concretely, the cell culture was carried out on a cultureplate using Dulbecco's minimum essential medium [manufactured byGibco-BRL] under the conditions of 5% CO₂, 20% O₂ atmosphere. The cellswere maintained at a density of 10³ cells/ml to 10⁶ cells/ml, and theexchange of the media and the dilution of the cells (with a freshmedium) were carried out once in two days.

As a result, as is observed in rad51b (indicated by RAD51B-/-) mutant,the proliferation rates of rad51c mutant, rad51d mutant, xrcc2 mutantand xrcc3 mutant were significantly lower than that of wild-type cells.While the length of the cell cycle is comparable between wild-type andmutant clones, higher proportions of dead cells were seen in thesemutant clone cultures as shown in FIG. 1. Therefore, it was demonstratedthat the rad51 mutant clones showed slower growth rates.

EXAMPLE 2 Chromosome Analysis

(1) Chromosome Analysis

In order to investigate the cause of cell death, chromosome analysis ofmetaphase-arrested cells was performed. The results are shown in Table1.

TABLE 1 Spontaneous Chromosomal Aberrations Genotypes of Chromosome-typeChromatid-type Total Total Cells Analyzed Breakages Gaps Breakages GapsBreakages + Gaps Breakages Wild-type 0.25 0.25 0.25 1.25   2 ± 0.7 0.5 ±0.5 rad51c 0 6 6.7 3.3 16.0 ± 3.3 6.7 ± 2.1 rad51d 14 8.7 10 6 38.7 ±5.1  24 ± 4.0 xrcc2 0 9.3 6.7 5.3 21.0 ± 3.8 6.7 ± 2.1 xrcc3 2.7 10.76.7 6 26.0 ± 4.2 9.4 ± 2.5

In Table 1, data are presented as the number of aberrations per 100cells. At least 150 mitotic cells were analyzed for each genotype. Totalaberrations per cell and SE were calculated as follows: Concretely, thecells are treated with colcemid for 3 hours, and then fixed. On thefollowing day, the fixed cells are fixed on a slide glass and thenstained. One-hundred or more cells in which the number of chromosomes isnormal and the chromosomes are condensed were analyzed, and the numberof chromosomal breakages was counted.

It is found from the results of Table 1 that in the four kinds of themutant clones, the levels of spontaneous chromosomal breakages, whichare causative for the reduced viability, are significantly increased.These findings are considerably consistent with those for rad51b DT40cells, and qualitatively similar to those for XRCC2-deficient hamstercells and XRCC3-deficient hamster cells [the above-mentioned Tebbs etal. (1995); the above-mentioned Cartwright et al., Nucleic Acids Res.,26, 3084–3089 (1998); the above-mentioned Liu et al. (1998)].

The spontaneous chromosomal aberrations in each of these mutant clonecultures obtained in Example 1 mentioned above could be caused bydefective homologous recombination repair of replication-associateddouble-strand DNA breakages [the above-mentioned Haber (1999)]. In orderto evaluate the homologous recombination capacity of each mutant, boththe efficiency of targeted integration of transfected genomic DNAfragments and the level of sister chromatid exchange were determined.

(2) Determination of Targeted Integration Frequencies

In order to analyze targeted integration events at the β-actin,ovalbumin [the above-mentioned Buerstedde et al. (1991)] and XRCC2 loci,Southern blot analysis was carried out for each of the mutant clonesobtained in Example 1 mentioned above.

The gene targeting construct obtained in the Example 1 mentioned abovewas transfected into the wild-type DT40 cells and each mutant cell, anda clone resistant to an appropriate antibiotic was sorted out. Each ofchromosomal DNAs was isolated from each of the resulting clones by aconventional method. Next, Southern blot analysis was carried out foreach of the chromosomal DNA using a DNA fragment (a size of up to 1 kb)just near the gene targeting construct in each locus as a probe. Thehybridization conditions were ordinary conditions, i.e. hybridization:65° C., 1 M NaCl, 50 mM Tris-HCl (pH 7.4), 0.5% SDS, Denhardt'ssolution, denatured salmon sperm DNA (10 μg/ml); washing: 65° C.,0.3×SSC, 0.1% SDS.

Thereafter, the targeted integration frequency was evaluated by thefollowing formula:[the number of colonies showing a band having a pattern different fromthe band of the parent strain, each of the bands being shown by Southernblotting]/[the number of total drug resistance colonies analyzed]The results are shown in Table 2.

TABLE 2 Targeted Integration Frequencies Targeting Constructs GenotypeXRCC2-puro *Ov-puro *Ov-neo KU70-hyg Wild-type 12/20 (60%) 24/37 (65%)18/36 (50%) 11/18 (61%) rad51c   2/48 (4.2%)  5/46 (11%) ND ND rad51d0/38 0/22 ND ND xrcc2 ND ND 0/34 0/43 xrcc3 0/42 0/36 ND ND In thetable, data show the number of targeted clones in each locus per numberof drug resistance clones analyzed. Also, in the table, the ratio oftargeted integration events is given in parenthesis. Further, in thetable, two targeting constructs of the ovalbumin locus comprise either apuromycin resistance gene or a neomycin resistance gene. ND means notdeterminable.

As shown in Table 2, the targeted integration frequencies were reducedby about 8-fold in the rad51c clone and by at least 30-fold in therad51d clone, the xrcc2 clone and the xrcc3 clone.

(3) Analyses of Chromosome Aberrations and Sister Chromatid Exchange

Analyses of chromosome and sister chromatid exchange were carried out inaccordance with the descriptions given in the above-mentioned Sonoda etal. (1998) and the above-mentioned Sonoda et al. (1999). Concretely,living cells are cultured in the presence of BrdU for double celldivision cycles. After performing the manipulation of rupture andfixation of the cells by hypotonic fixation, the cells (chromosomes) arefixed on a slide glass. The fixed cells are irradiated with ultravioletrays to break genomic DNA resulting from incorporation of BrdU into boththe double strand. The protein adherent to the broken DNA is washedaway, and the remaining chromatin protein adherent to genomic DNA isstained.

In order to analyze MMC-inducing sister chromatid exchanges, cells wereincubated in a medium containing 0.05 μg/ml MMC in an atmosphere of 5%CO₂ and 20% O₂, at humidity of 100%, and at 39.5° C. (or may be 37° C.)for 12 hours (the length of the cell cycle of DT40 cells being about 8hours). Colcemid (N-deacetyl-N-methylcolchicine) was added in aconcentration of 0.1 mg/ml at a point 1.5 hours before the harvest ofthe cells, and the mixture was further incubated. The results for eachof the sister chromatid exchanges are shown in FIG. 2.

It is seen from FIG. 2 that the Rad51-paralog mutants exhibitsignificantly reduced levels of both the spontaneous sister chromatidexchanges as well as the sister chromatid exchanges induced by mitomycinC (MMC), a cross-linking agent. Also, the present inventors havepreviously shown that the sister chromatid exchange events would reflectpost-replicational repair by homologous recombination that is probablyassociated with crossing-over between sister duplexes [theabove-mentioned Sonoda et al. (1999)]. These findings suggest that eachparalog is actually involved in the homologous recombination.

EXAMPLE 3

(1) Sensitivity of Cells Against DNA-Damaging Factors (γ-Rays, MMC andCisplatin)

Cells which were serially diluted (10²×10 cells/μl, 10³×10 cells/μl,10⁴×10 cells/μl and 10⁵×10 cells/μl) were plated on a plate of methylcellulose-containing medium [composition: Dulbecco's medium, fetalbovine serum (15%), chicken serum (1.5%), methyl cellulose (15 g/l)],and cultured in an atmosphere of 5% CO₂ and 20% O₂, at humidity of 100%,and 39° C. Next, ¹³⁷Cs γ-ray source was irradiated to the plate for 30to 90 seconds. Further, the cells were cultured in an atmosphere of 5%CO₂ and 20% O₂, at humidity of 100%, and 39° C. for one week.Thereafter, colony survival assay was carried out using the number ofcolonies on a plate without subjected to γ-ray irradiation as control,thereby evaluating sensitivity of the cells to γ-ray irradiation.

In addition, the cells were incubated in a complete medium containingMMC (manufactured by KYOWA HAKKO KOGYO CO., LTD.) at 39° C. for 1 hour,and washed with a warm medium at 37° C. thrice. The washed cells werethen plated on the above-mentioned methyl cellulose-containing medium.The cells were cultured in an atmosphere of 5% CO₂ and 20% O₂, athumidity of 100%, and 39° C. Thereafter, the colony survival assay wascarried out using the number of colonies appearing on the plate on whichthe cells untreated with MMC were plated as control, thereby evaluatingsensitivity of the cells to MMC.

Further, the cells were plated on a plate of methyl cellulose containingcisplatin (manufactured by NIPPON KAYAKU CO., LTD.), and cultured in anatmosphere of 5% CO₂ and 20% O₂, at humidity of 100%, and 39° C.Thereafter, the colony survival assay was carried out, therebyevaluating sensitivity of the cells to cisplatin.

The biologically associated DNA repair capacity of each mutant wasevaluated in colony survival assays after exposure to a DNA-damagingfactor. As a result, clearly, rad51c mutant, rad51d mutant, xrcc2 mutantand xrcc3 mutant all showed a very similar pattern of sensitivities,which were also consistent with those of rad51b cells. As shown in thepanel (A) of FIG. 3, the γ-ray sensitivity of each mutant was mild, eachof which was about 3-fold sensitive to MMC than that of the wild-typeDT40 cells, based on estimated D10 values.

However, as shown in the panel (B) of FIG. 3, each mutant was about8-fold more sensitive than normal cells to killing by cisplatin(cis-diamminedichloroplatinum-II), a DNA cross-linking agent that hasbeen widely used in chemotherapy. The complementation of these mutantswith human cDNAs corresponding to mutant genes in these mutants restoredtheir cisplatin sensitivity to nearly normal levels.

From these results, it is confirmed that the rad51 paralog genedisruptions were causation for the increased sensitivity to cisplatin.It should be noted that the previous findings on XRCC2 and XRCC3 mutanthamster cells [the above mentioned Tebbs et al. (1995); the abovementioned Liu et al. (1998)] are considerably consistent with the newcorresponding DT40 mutants of the present inventors for all propertiesexamined (mild radiosensitivity, high sensitivity to cross-linkingagents, chromosomal instability, and defective homologous recombination)except that the mutant hamster cells showed more pronounced sensitivityto MMC, i.e., only a few-fold sensitive to γ-rays while 60 to 70-foldsensitive to MMC based on estimated D10 values [the above-mentionedThompson et al. (1999)]. Accordingly, the role of the Rad51 paralogs inthe homologous recombination repair is likely to be conserved betweenDT40 and mammalian cells.

(2) Phenotypic Suppression of Rad51-Paralog Mutants by Rad51Overexpression

In yeast, it has been shown that the overexpression of Rad51 partiallysuppresses the ionizing radiation sensitivity of rad55 and rad57 mutantstrains [the above-mentioned Hays et al. (1995); the above-mentionedJohnson et al. (1995)]. Furthermore, it has been shown that theoverexpression of human Rad51 cDNA in rad51b cells also restores thesensitivity to γ-rays and MMC (but not restoring to cisplatin) towild-type levels.

Similar to these, as shown in the panel (B) of FIG. 3, Rad51overexpression partially complements the sensitivities of each of rad51cmutant, rad51d mutant, xrcc2 mutant and xrcc3 mutant to cisplatin.Accordingly, it is seen that human Rad51 at least partially complementsfor each of these paralogs under conditions where the amount of humanRad51 protein is highly overexpressed as compared with the endogenousRad51 level [the panel (C) of FIG. 3].

EXAMPLE 4 Analysis of Nuclear Rad51 Focus Formation

In order to further evaluate the role of the paralogs in the homologousrecombination, the nuclear Rad51 focus formation was analyzed by γ-rayirradiation.

Foci that can be microscopically confirmed show an assembly of Rad51into atypically long nucleoprotein filaments probably involved inhomologous recombination repair [the above-mentioned Bishop et al.(1998); Raderschall, E. et al., Proc. Natl. Acad. Sci. USA, 96,1921–1926 (1999)].

Therefore, wild-type and mutant cultures with progressing cell cycleswere exposed to γ-rays and MMC, and thereafter the cells wereimmunostained with anti-Rad51 antiserum. The results are shown in FIG.4.

It is seen from FIG. 4 that the formation of Rad51 foci was severelyimpaired in each mutant cell line after ionizing radiation and MMCtreatments, as previously observed in XRCC3-deficient hamster cells [theabove-mentioned Bishop et al. (1998)]. After five hours of the ionizingradiation or MMC treatment, less than 15% of the mutant cells containeda significant number of distinct Rad51 foci (exceeding 4 per cell),whereas more than 60% of wild-type cells showed robust formation of thefoci. Since protein levels of Rad51 did not change after genotoxictreatment in any mutant clone (data not shown), these results show thatall Rad51 paralogs are required for damage-induced redistribution ofRad51 within the nucleus. In the absence of each of these five kinds ofthe paralog proteins, there must still be a basal level of Rad51activity since the mutants are viable. Accordingly, these five kinds ofthe proteins are required in the formation of very long-track Rad51filaments, which are microscopically visible.

The reconstruction of Rad51 focus formation could not be evaluatedbecause of high background level of immunostaining in the presence ofoverexpressed human Rad51 protein. These data combined with thedefective Rad51 focus formation in every Rad51-paralog mutant suggestthat all these proteins are involved in the recruitment of Rad51 intonucleoprotein filaments that mediate homologous pairing and exchange.

INDUSTRIAL APPLICABILITY

According to the present invention, there is enabled a screening of anovel anticancer agent which allows a more efficient therapy for acancer, wherein the agent is an agent capable of enhancing thesensitivity of a cell to an anticancer agent comprising a DNA-damagingfactor or an agent having a DNA-damaging action.

1. An isolated human mutant Rad51 paralog nucleic acid moleculecomprising SEQ ID NO: 1, except that nucleotides 463–573 of SEQ ID NO: 1are deleted or substituted with a marker gene, wherein the marker geneis selected from the group consisting of puromycin resistance gene,histidinol resistance gene, Ecogpt gene, blastocitidin resistance geneand hygromycin resistance gene.
 2. The isolated human mutant Rad51paralog nucleic acid molecule according to claim 1, wherein a proteinencoded thereby shows an activity for enhancing sensitivity of a cell toa DNA damaging factor, and wherein the DNA-damaging factor is selectedfrom one or more of the group consisting of: irradiation rays, mitomycinC and cisplatin.
 3. An isolated transformed cell having the nucleic acidmolecule of claim
 1. 4. The isolated transformed cell according to claim3, wherein the cell is DT40.
 5. A screening method for a test substancehaving DNA damaging action comprising the steps of: a) contacting a testsubstance with the transformed cell of claim 3 or 4; and b) evaluating aresponse of the cell obtained in step a), wherein the response of thecell is evaluated by analyzing cell growth rate, chromosomal breakages,homologous recombination, or sister chromatid exchange as an indicatorof DNA damaging action of the test substance.